Modulation of Pain Signaling Mechanisms by botulinum neurotoxin A Noxious pain occurs in several diseases and also manifests as a disease by itself. Pain affects more humans than diabetes, heart diseases and cancer combined. Currently available therapeutic strategies that use narcotic and non-narcotic medications have limitations due to the adverse reactions and toxicities associated with the therapy. Treatment of chronic pain with narcotic analgesics leads to the dependence of their use resulting in increased tolerance and addiction. This is a major health hazard in the United States. Therefore, developing novel strategies that alleviate pain without any adverse reactions is very important and it forms the basis of our long-term goal. Recent research and clinical investigations suggest the potential of botulinum neurotoxin A (BTX-A) as an emerging alternative to treat chronic pain conditions without any use dependence or severe adverse reactions. Injection of BTX-A has been shown to produce a faster and long-lasting relief from chronic pain associated with peripheral vascular diseases and several forms of sensory pain due to nerve damage or injury. Although BTX-A is very effective in treating pain, the mechanism underlying its analgesic action still remain unknown. Therefore, the immediate goal of this research proposal is to fill this knowledge gap. We hypothesize that ?BTX-A inhibits pain by inhibiting the sensitization of transient receptor potential vanilloid 1 (TRPV1) / Ankyrin 1 (TRPA1) channel proteins that are implicated in pain sensation. This is novel as we propose to analyze a SNARE protein independent effect of BTX-A on TRPV1/TRPA1 channel proteins. We propose two specific aims which will evaluate the effects of BTX-A on sensitization of TRPV1/TRPA1 channel proteins by PKC and examine the effects of BTX-A magnetic nanoparticles (BMN), delivered and trapped to specific target sites by external magnetic field (EMaF), to produce long-lasting pain relief. To achieve our research objectives we will employ electrophysiological, molecular biological and biochemical experiments and use whole animal models where pain will be induced by chemical or chronic constriction injury. We will isolate dorsal root ganglion (DRG) neurons from wild type and TRPV1-/-/TRPA1-/- mice to determine the effects of BTX-A on their sensitization by protein kinase C. Alternative to DRG neurons, we will use HEK293 cells that stably express TRPV1/TRPA1. Collectively, the outcomes of this study will 1). Advance our knowledge on a novel TRPV1/TRPA1-dependent mechanism by which BTX-A inhibits pain sensitization. 2). Provide a target site-specific polymer coated drug delivery system for BTX-A nanoparticles to produce a site-specific analgesic effect. This will advance our long term-goal of finding a new therapeutic strategy to treat pain.